Starch mobilization and sucrose accumulation in the pulp of Keitt mangoes during postharvest ripening

Sugar is a determinant for the quality of mangoes, but information about its accumulation is scarce. Although starch can contribute to sugar production during ripening, not much is known about the enzymes involved. This work presents the changes in carbohydrate and enzymes during the development and ripening of Keitt mangoes. Starch disappearance was concomitant to a fivefold increase of sucrose, the most abundant sugar of the ripe fruits. The activities of alpha-amylase, beta-amylase, phosphorylase and isoamylase were detected in the pulp, and while alpha-amylase increased parallel to the starch content, beta-amylase presented a 20-fold increase during ripening. On the other hand, high phosphorylase activity was observed when fruits were still accumulating starch, and lowered during ripening. Isoamylase was detected during development and increased slightly during ripening, which would be in agreement to the expected role for isoamylases as acting on both subproduct of starch synthesis and degradation.

Effects of Cold Stress, Corticosterone and Catecholamines on Phagocytosis in Mice: Differences between Resting and Activated Macrophages

Objective: We subjected mice to acute cold stress and studied the effect on phagocytosis by peritoneal macrophages mediated by 3 types of phagocytic receptors: Fc gamma, complement receptors 3 (CR3) and mannose and beta-glucan receptors. Methods: Mice were subjected to a cold stress condition (4 C for 4 h), and then peritoneal macrophages were harvested and phagocytosis assays performed in vitro. Results: We found a striking difference between resting and lipopolysaccharide (LPS)-activated macrophages (by intraperitoneal injection of LPS 4 days before the stress experiment): for resting macrophages cold stress caused a decrease in phagocytosis mediated by Fc gamma or mannose receptors, while for activated macrophages we observed an increase in phagocytosis by the 3 types of receptors. These effects were associated with an increase in plasma concentrations of corticosterone and catecholamines following the cold stress. In order to verify whether these hormone changes could account for the observed effects on phagocytosis, we performed in vitro assays by incubating macrophages harvested from nonstressed animals with these hormones for 4 h at 37 degrees C and measuring their phagocytic capacity. The following experiments were done: (a) with resting (nonactivated) macrophages; (b) with macrophages previously activated in vitro by incubation with LPS; (c) with macrophages previously activated in vivo by intraperitoneal injection of mice with LPS, 4 days before harvesting the cells. We found that for resting macrophages, corticosterone decreased phagocytosis mediated by Fc gamma and mannose and beta-glucan receptors, but catecholamines had no effect. For macrophages activated either in vivo or in vitro, catecholamines caused an increase in phagocytosis (excluding mannose receptors) while corticosterone had no effect. Conclusion: The above findings suggest that stress can regulate phagocytosis in different ways, depending on the kind of phagocytic receptor involved, the level of stress hormones and the physiological state of the macrophages. Copyright (C) 2010 S. Karger AG, Basel

Isolation of transcripts overexpressed in the human pathogen Trichophyton rubrum grown in lipid as carbon source

Trichophyton rubrum is the most common etiological agent of human dermatophytosis. Despite the incidence and medical importance of this dermatophyte, little is known about the mechanisms of host invasion and pathogenicity. Host invasion depends on the adaptive cellular responses of the pathogen that allow it to penetrate the skin layers, which are mainly composed of proteins and lipids. In this study, we used suppression subtractive hybridization to identify transcripts over-expressed in T rubrum cultured in lipid as carbon source. Among the subtractive cDNA clones isolated, 85 clones were positively screened by cDNA array dot blotting and were sequenced. The putative proteins encoded by the isolated transcripts showed similarities to fungal proteins involved in metabolism, signaling, defense, and virulence, such as the MDR/ABC transporter, glucan 1,3-beta-glucosidase, chitin synthase B, copper-sulfate-regulated protein, and serine/threonine phosphatase (calcineurin A). These results provide the first molecular insight into the genes differentially expressed during the adaptation of T. rubrum to a lipidic carbon source.

Prevention of Salmonella Typhimurium colonization and organ invasion by combination treatment in broiler chicks

The effects in broiler chicks of treatment with a competitive exclusion (CE) product, an experimental dietary probiotic, and the abiotic beta-glucan on cecal colonization, organ invasion, and serum and intestinal IgG and IgA levels to Salmonella challenge was evaluated. Four groups of 1-d-old chicks were treated by oral gavage on d 1 with an appropriate dose of a commercial CE product. Three groups received daily doses of probiotic, beta-glucan, or both, for 6 d. Three other groups were fed daily from d 1 onwards with probiotic, beta-glucan, or both. Subgroups of 30 chicks from each group were challenged on d 1, 9, 16, or 23 with 10(7) cfu/mL of Salmonella Typhimurium (1769NR) and killed 7 d later. Control groups were maintained untreated and remained unchallenged (negative control), or were challenged with Salmonella Typhimurium (1769NR; positive control), as described above. Cecum, liver, and spleen samples were examined for the presence of Salmonella, whereas serum and intestinal fluid samples were assayed for total antibody (IgG and IgA) concentrations. Data were analyzed by 1-way ANOVA, and means were compared using Duncan`s multiple range test. In comparison with other treatments, those involving CE product and beta-glucan, with or without probiotic during the first week, resulted in a superior inhibition of cecal colonization and organ invasion by Salmonella and also offered a higher level of protection (P < 0.05). During the second week, treatments containing experimental dietary probiotic and beta-glucan, with or without CE product, resulted in an inhibition of liver invasion (P < 0.05). The IgA levels were significantly higher (P < 0.05) in intestinal fluid compared with serum, whereas IgG had low levels. The results in the first and third week indicate that combination treatments involving CE product, probiotic, and beta-glucan are a more effective control of Salmonella colonization than the corresponding individual preparations.

Polysaccharide fraction of Agaricus brasiliensis avoids tumor-induced IL-10 production and changes the microenvironment of subcutaneous Ehrlich adenocarcinoma

Subcutaneous Ehrlich tumor-bearing mice were treated with in situ inoculation of a P-glucan-rich extract of Agaricus brasiliensis (ATF), which reduced tumor growth. Histopathological analysis showed that the tumor masses of control mice (Ehr) presented giant tumor cells and many mitotic figures whereas the tumor tissue obtained from ATF-treated animals (Ehr-ATF) presented a lower frequency of both mitotic and giant cells, associated with a higher frequency of apoptotic cells than Ehr. Analysis of the lymphoproliferative activity of spleen cells showed that the treatment had a suppressive rather than a stimulatory effect. Spleen cells of the Ehr group produced higher in vitro levels of IL-10 than normal controls and this occurrence was partially avoided by treatment with ATF. Analysis of cytokine production by tumor-infiltrating cells (ELISpot) showed that ATF induced a higher number of IFN-gamma-producing cells at 7 and 14 days as well as reduction of IL-10-secreting cells at the latter time. Confocal microscopy analysis showed higher intensity of labeling of CD4+ and Mac-3+ cells in ATF-treated mice. Analysis of in situ expression of angiogenic growth factors showed a slight decrease of FGF-2 mRNA in Ehr-ATF animals (7th day) but not of VEGF-A or TGF-beta expression. This fraction could not directly lyse either lymphocytes or tumor cells and we speculate that antitumor effect of ATF could be due to induction of a selective migration of immunocompetent cells from the spleen to the tumor site and to the switch of cytokine production. (C) 2009 Elsevier Inc. All rights reserved.

Long-term metabolic and skeletal muscle adaptations to short-sprint training: Implications for sprint training and tapering

The adaptations of muscle to sprint training can be separated into metabolic and morphological changes. Enzyme adaptations represent a major metabolic adaptation to sprint training, with the enzymes of all three energy systems showing signs of adaptation to training and some evidence of a return to baseline levels with detraining. Myokinase and creatine phosphokinase have shown small increases as a result of short-sprint training in some studies and elite sprinters appear better able to rapidly breakdown phosphocreatine (PCr) than the sub-elite. No changes in these enzyme levels have been reported as a result of detraining. Similarly, glycolytic enzyme activity (notably lactate dehydrogenase, phosphofructokinase and glycogen phosphorylase) has been shown to increase after training consisting of either long (> 10-second) or short (< 10-second) sprints. Evidence suggests that these enzymes return to pre-training levels after somewhere between 7 weeks and 6 months of detraining. Mitochondrial enzyme activity also increases after sprint training, particularly when long sprints or short recovery between short sprints are used as the training stimulus. Morphological adaptations to sprint training include changes in muscle fibre type, sarcoplasmic reticulum, and fibre cross-sectional area. An appropriate sprint training programme could be expected to induce a shift toward type Ha muscle, increase muscle cross-sectional area and increase the sarcoplasmic reticulum volume to aid release of Ca2+. Training volume and/or frequency of sprint training in excess of what is optimal for an individual, however, will induce a shift toward slower muscle contractile characteristics. In contrast, detraining appears to shift the contractile characteristics towards type IIb, although muscle atrophy is also likely to occur. Muscle conduction velocity appears to be a potential non-invasive method of monitoring contractile changes in response to sprint training and detraining. In summary, adaptation to sprint training is clearly dependent on the duration of sprinting, recovery between repetitions, total volume and frequency of training bouts. These variables have profound effects on the metabolic, structural and performance adaptations from a sprint-training programme and these changes take a considerable period of time to return to baseline after a period of detraining. However, the complexity of the interaction between the aforementioned variables and training adaptation combined with individual differences is clearly disruptive to the transfer of knowledge and advice from laboratory to coach to athlete.

Antagonism of the two-needle pine stem rust fungi Cronartium flaccidum and Peridermium pini by Cladosporium tenuissimum in vitro and in planta

Selected isolates of Cladosporium tenuissimum were tested for their ability to inhibit in vitro aeciospore germination of the two-needle pine stem rusts Cronartium flaccidum and Peridermium pini and to suppress disease development in planta. The antagonistic fungus displayed a number of disease-suppressive mechanisms. Aeciospore germination on water agar slides was reduced at 12, 18, and 24 h when a conidial suspension (1.5 x 10(7) conidia per ml) of the Cladosporium tenuissimum isolates was added. When the aeciospores were incubated in same-strength conidial suspensions for 1, 11, 21, and 31 days, viability was reduced at 20 and 4 degreesC. Light and scanning electron microscopy showed that rust spores were directly parasitized by Cladosporium tenuissimum and that the antagonist had evolved several strategies to breach the spore wail and gain access to the underlying tissues. Penetration occurred with or without appressoria. The hyperparasite exerted a mechanical force to destroy the spore structures (spinules, cell wall) by direct contact, penetrated the aeciospores and subsequently proliferated within them. However, an enzymatic action could also be involved. This was shown by the dissolution of the host tell wall that comes in contact with the mycelium of the mycoparasite, by the lack of indentation in the host wall at the contact site, and by the minimal swelling at the infecting hyphal tip. Culture filtrates of the hyperparasite inhibited germination of rust propagules. A compound purified from the filtrates was characterized by chemical and spectroscopic analysis as cladosporol, a known beta -1,3-glucan biosynthesis inhibitor. Conidia of Cladosporium tenuissimum reduced rust development on new infected pine seedlings over 2 years under greenhouse conditions. Because the fungus is an aggressive mycoparasite, produces fungicidal metabolites, and can survive and multiply in forest ecosystems without rusts, it seems a promising agent for the biological control of pine stem rusts in Europe.

Starch degradation in 'Kensington' mango fruit following heat treatments

Changes in carbohydrate metabolism of 'Kensington' mango fruit from 2 major production regions in Queensland were measured after conditioning fruit with hot air at 40degreesC for 0, 2, 4, 8 and 16 h or at 22degreesC for 16 h (control) followed by hot-water treatment at either 45degreesC fruit-core temperature for 30 min or 47degreesC fruit-core temperature held for 15 min. Advancing physiological maturity of 'Kensington' mango fruit was correlated with increased starch concentration within the mesocarp. An alpha-amylase inhibitor was present in unripe 'Kensington' mesocarp. alpha-Amylase activity was promoted by conditioning fruit at 40degreesC for 8 h, and this enhanced enzyme activity persisted until the fruit were ripe. Consequently, starch degradation was accelerated and the concentration of total soluble solids was higher in fruit conditioned at 40degreesC for 8 h than in fruit left at the lower temperature of 22degreesC for 16 h or not conditioned. Immediately on removal of fruit from hot-water treatment, activities of alpha-amylase and phosphorylase were inhibited. This inhibition was correlated with higher starch concentration and starch layer and starch spot injuries in these fruit. A positive correlation was also found between increased sucrose concentration and greater starch loss in 40degreesC conditioned 'Kensington' fruit. It is proposed that increased sugar concentration in the mesocarp increased the level of fruit heat tolerance.

A high throughput colorimetric assay of beta-1,3-D-glucans by Congo red dye

Mushroom strains contain complex nutritional biomolecules with a wide spectrum of therapeutic and prophylactic properties. Among these compounds, β-d-glucans play an important role in immuno-modulating and anti-tumor activities. The present work involves a novel colorimetric assay method for β-1,3-d-glucans with a triple helix tertiary structure by using Congo red. The specific interaction that occurs between Congo red and β-1,3-d-glucan was detected by bathochromic shift from 488 to 516 nm (> 20 nm) in UV–Vis spectrophotometer. A micro- and high throughput method based on a 96-well microtiter plate was devised which presents several advantages over the published methods since it requires only 1.51 μg of polysaccharides in samples, greater sensitivity, speed, assay of many samples and very cheap. β-d-Glucans of several mushrooms (i.e., Coriolus versicolor, Ganoderma lucidum, Pleurotus ostreatus, Ganoderma carnosum, Hericium erinaceus, Lentinula edodes, Inonotus obliquus, Auricularia auricular, Polyporus umbellatus, Cordyseps sinensis, Agaricus blazei, Poria cocos) were isolated by using a sequence of several extractions with cold and boiling water, acidic and alkaline conditions and quantified by this microtiter plate method. FTIR spectroscopy was used to study the structural features of β-1,3-d-glucans in these mushroom samples as well as the specific interaction of these polysaccharides with Congo red. The effect of NaOH on triple helix conformation of β-1,3-d-glucans was investigated in several mushroom species.

A novel colorimetric assay of beta-D-glucans in basidiomycete strains by alcian blue dye in a 96-well microtiter plate

Basidiomycete strains synthesize several types of beta-D-glucans, which play a major role in the medicinal properties of mushrooms. Therefore, the specific quantification of these beta-D-glucans in mushroom strains is of great biochemical importance. Because published assay methods for these beta-D-glucans present some disadvantages, a novel colorimetric assay method for beta-D-glucan with alcian blue dye was developed. The complex formation was detected by following the decrease in absorbance in the range of 620 nm and by hypsochromic shift from 620 to 606 nm (similar to 14 nm) in UV-Vis spectrophotometer. Analysis of variance was used for optimization of the slope of the calibration curve by using the assay mixture containing 0.017% (w/v) alcian blue in 2% (v/v) acetic acid at pH 3.0. The high-throughput colorimetric assay method on microtiter plates was used for quantification of beta-D-glucans in the range of 0-0.8 mu g, with a slope of 44.15 x 10(-2) and a limit of detection of 0.017 mu g/well. Recovery experiments were carried out by using a sample of Hericium erinaceus, which exhibited a recovery of 95.8% for beta-1,3-D-glucan. The present assay method exhibited a 10-fold higher sensitivity and a 59-fold lower limit of detection compared with the published method with congo red beta-D-glucans of several mushrooms strains were isolated from fruiting bodies and mycelia, and they were quantified by this assay method. This assay method is fast, specific, simple, and it can be used to quantify beta-D-glucans from other biological sources. (C) 2015 American Institute of Chemical Engineers

Generation of high-affinity monoclonal antibodies of IgG class against native β-d-glucans from basidiomycete mushrooms

β-d-glucans from basidiomycete strains are powerful immunomodulatory agents in several clinical conditions. Therefore, their assay, purification and characterization are of great interest to understand their structure-function relationship. Hybridoma cell fusion was used to raise monoclonal antibodies (Mabs) against extracellular β-d-glucans (EBGs) from Pleurotus ostreatus. Two of the hybridoma clones (1E6-1E8-B5 and 3E8-3B4) secreting Mabs against EBGs were selected. This hybridoma cell line secreted Mabs of the IgG class which were then purified by hydroxyapatite chromatography to apparent homogeneity on native and SDS-PAGE. Mabs secreted by 1E6-1E8-B5 clone were found to recognize a common epitope on several β-d-glucans from different basidiomycete strains. This Mab exhibited high affinity constant (KA) for β-d-glucans from several mushroom strains in the range of 3.20 × 109 ± 3.32 × 103-1.51 × 1013 ± 3.58 × 107 L/mol. Moreover, they reacted to some heat-treated β-d-glucans in a different mode when compared with the native forms; these data suggest that this Mab binds to a conformational epitope on the β-d-glucan molecule. The epitope-binding studies of Mabs obtained from 1E6-1E8-B5 and 3E8-3B4 revealed that the Mabs bind to the same epitope on some β-d-glucans and to different epitopes in other antigen molecules. Therefore, these Mabs can be used to assay for β-d-glucan from basidiomycete mushrooms. © 2015 Elsevier Ltd. All rights reserved.

Human mucocutaneous leishmaniasis in Três Braços, Bahia - Brazil: an area of Leishmania braziliensis braziliensis transmission. I. Laboratory diagnosis

Leishmanial parasites were detected in 71.2% of patients with cutaneous disease and 48% of patients with mucosal disease, using principally scanning of imprints mears and histological sections and hamster inoculation. Parasites were more frequent in early cutaneous lesions (p < 0.005) o fless than two month duration. Also they were more common in multiple than single mucosal lesions (p < 0.02) in spite of considerable prior glucan time therapy in the former group. 93% of cutaneous lesions had a positive leishmanin skin test and most of the negatives occurred in patients with lesions of less than one month duration. 97% of patients with single mucosal lesion and 79% with multiple mucosal lesions had a positive skin test. 86% of cutaneous disease and 90% of mucosal disease was associated with a positive indirect immunofluorescent antibody test at a ≥ 1/20 dilution. In both groups multiple lesions were associated with higher titres and titres were significantly higher in patients with mucosal disease compared with cutaneous disease (p < 0.01).

Interplay of Exoribonucleases, Hfq and Small RNAs Structural Determinants in the Control of Gene Expression

Dissertation presented to obtain the Ph.D degree in Biology

Chemical genetic analysis of cdc2p : new insights into the regulation of cytokinesis in "Schizosaccharomyces pombe"

RÉSUMÉ La protéine kinase cyciine-cdc2p (Cdk) joue un rôle fondamental dans la progression du cycle cellulaire dans la levure de fission Schizosaccharomyces pombe. Nous avons étudié le rôle de cdc2p dans la régulation de la cascade de septation ou SIN (septation initiation network) en mitose et en méiose. Le SIN contrôle l'initiation de la cytokinèse à la fin de la mitose, et est supposé être négativement régulé par cdc2p. Nous avons mutagénéisé le site actif de cdc2p afin qu'il puisse lier un analogue de l'ATP (PP1) qui agit comme inhibiteur. Cet analogue ne peut pas lier la kinase de type sauvage. Cette approche dite «chemical genetics» permet une meilleure résolution temporelle comparée à l'approche classique utilisant les mutants sensibles à une température élevée. Nous avons montré que ce mutant cdc2-as (analogue sensitive) est fonctionnel et que, in vitro, l'activité kinase est inhibée en présence de l'analogue. Les cellules portant cette mutation, contrairement aux cellules de type sauvage s'arrêtent de manière irréversible soit en G2 soit en G1 et G2, suivant la concentration de l'inhibiteur. L'inactivation de cdc2p-as dans des cellules arrêtées en métaphase conduit au recrutement asymétrique des protéines du SIN sur le pôle du fuseau mitotique et au recrutement des composants du SIN, ainsi que de la ß-(1,3)glucan synthase à l'anneau contractile. De plus, nos résultats montrent que l'orthologue de la phosphatase cdc14p dans S. pombe, fip1p/clp1p, joue un rôle dans la régulation de la localisation des protéines du SIN suite à l'inactivation de cdc2p. Finalement, l'activité de cdc2p est requise pour maintenir la polo-like kinase plo1p sur les pôles du fuseau mitotique dans les premiers stages de la mitose. C'est pourquoi nous concluons que l'inactivation de cdc2p est suffisante pour activer le SIN et promouvoir la cytokinèse. Dans une étude séparée, nous avons caractérisé des potentiellement nouveaux composants ou régulateurs du SIN qui ont été isolés dans deux criblages génétiques visant à isoler des mutants atténuants la signalisation du SIN. Summary : The cyclin dependent protein kinase (Cdk) cdc2p plays a central role in the cell cycle progression of fission yeast Schizosaccharomyces pombe. We have studied the role of cdc2p in regulating the septation initiation network (SIN) in mitosis and meiosis. The SIN regulates the initiation of cytokinesis at the end of mitosis and is thought to be inhibited by cdc2p. We have mutated the active site of cdc2p to permit binding of an inhibitory ATP analogue (PP1), which is unable to bind unmodified kinases. This "chemical genetic" approach provides a much higher temporal resolution than it can be achieved with classical temperature-sensitive mutants. We demonstrate that cdc2-as (analogue sensitive) is functional and that addition of PP1 inhibits cdc2p kinase activity in vitro. Cells carrying the cdc2-as allele, but not cdc2+, undergo reversible cell cycle arrest following addition of PP1 either in G2, or at both major commitment points in the cell cycle (G1 and G2), depending upon the concentration of PP1. Inactivation of cdc2p-as in cells arrested in early mitosis promotes both the asymmetric recruitment of SIN proteins to the spindle pole bodies (SPBs), and the recruitment of the most downstream SIN components and ß-(1,3)-glucan synthase to the contractile ring. Furthermore, our results indicate that the S. pombe orthologue of Cdc14p, flp1p/clp1p, plays a role in regulating the relocalisation of SIN proteins following inactivation of cdc2p, and that cdc2p activity is required to retain the polo like kinase plot p on the SPBs in early mitosis. Thus, we conclude that inactivation of cdc2p is sufficient to activate the SIN and to promote cytokinesis. In a separate study, we have initially characterised potential novel components or regulators of the SIN pathway identified by two genetic screens for mutants attenuating SIN signaling.

Síntesi estereoselectiva d'anàlegs ciclobutènics d'L-nucleòsids

En els darrers 30 anys, els anàlegs de nucleòsids han estat una part essencial de la teràpia antiviral. Més recentment, els anàlegs carbocíclics de nucleòsids s'han convertit en importants objectius pel desenvolupament de nous agents terapèutics antivirals i antitumorals, en tant que l'absència de l'enllaç N-glicosídic els confereix una major estabilitat davant l'acció de les fosforilases. Per altra banda, s'ha descrit que alguns nucleòsids de configuració L presenten, en alguns casos, una bona activitat antiviral, una major estabilitat metabòlica i una toxicitat inferior a la dels seus homòlegs de configuració natural. El present treball planteja la síntesi estereoselectiva de derivats ciclobutènics de L-nucleòsids com a agents terapèutics, susceptibles de presentar una major activitat antiviral i una menor toxicitat que els agents actuals. Per assolir aquest objectiu, s'ha construït l'anell ciclobutènic mitjançant una reacció de fotocicloaddició [2+2]. Al mateix temps, s'ha desenvolupat un estudi de la influència del dissolvent en la reacció de fotocicloaddició [2+2] d'enones a alquens halogenats. A més, s'han estudiat diverses condicions de treball per dur a terme la reacció de deshalogenació dels derivats clorats preparats amb la metodologia anterior, utilitzant Zn com a reductor i amb un sistema d'escalfament per microones com a substituent dels mètodes d'escalfament convencionals. Aquest estudi ha permès disminuir notablement el temps d'aquesta reacció, passant de 7 hores a 20 minuts. Les condicions òptimes d'ambdues reaccions determinades amb aquests estudis han permès preparar l'intermedi clau per a la introducció de les bases nitrogenades, essent aquest un potencial precursor dels anàlegs ciclobutènics de nucleòsids, així com sintetitzar el primer producte de la ruta sintètica dissenyada que presenta la base nitrogenada a la seva estructura.